red = needs attention

italics = drafty language

blue = expanded information for the User Guide (remove for the quick-start guide)

I. Introduction

II. Procedure

A. Sample preparation and loading the sample

Turning on the SEM

1. Push the ‘power switch’ on the left-hand side of the microscope (Figure 1.2).  IMAGE GOES HERE 
2. Launch the Nanoeye software icon (Figure 2) on the desktop to launch the program IMAGE GOES HERE 
3. Prepare your sample for SEM analysis. Depending on the size and material, this may require gold/palladium sputter coating or carbon-coating (used for EDS). For sputter/carbon coater instructions, see the Sputter Coater Quick Start Guide.
   
   1. Common sample types include: stubs, with or without sputter coating; thin-sections with carbon coating; grain-mounts with carbon coating.
   2. Somewhere need to explain that coating is to prevent charging, which is especially common with thin-sections. Show a picture
4. Using the jig (Figure 3), measure the size of the specimen including mount. Use the gradations of the horizontal grid on the jig to measure the diameter of the specimen and the vertical grid for the specimen height. If you are using older 3.2mm style stubs with a narrow attachment post, there is an adapter available to fit into the stage. Make sure to measure dimensions with the adapter attached.IMAGE GOES HERE  
5. Important: use compressed air to ensure that blow off any loose material on the sample, that otherwise could be mobilized within the vacuum and damage the detectors.

Loading the sample into the SEM

1. When not in use, the SEM is typically left under vacuum. Thus, to load a sample, the SEM needs to be pumped with air. Press the Exchange button on the front of the SEM. The button LED light indicates the inner vacuum status of the chamber, and the signals are: 
   1. Light Off: Vacuum is not applied
   2. Light On: Vacuum is applied
   3. Slow blinking: Vacuum is being released
   4. Fast blinking:  Vacuum is being applied
2. Follow the LED strip on the front of the SEM which is a progress bar, with a fully illuminating LED strip indicating that the SEM is at vacuum. Listen for a double? beep, which indicates that the vacuum has fully been released.
3. Fully open the door and click the ‘Calibration’ button to initiate the stage motor calibration (Figure 4). This should take around 3 minutes, and all motor controls will go back to the home position when done and no numbers should be yellow.? Occasionally the motor will get stuck at its limit switch and the value will stay yellow, in which case you can run the Calibration routine again.
4. In the Nanoeye software, click the “Sample Information” text box (Figure 4, red rectangle), enter the height and width values, then press Enter. IMAGE GOES HERE  Important: The command will not be registered if the user fails to hit enter. After the height is entered, the Z-axis will automatically lower the stage to accommodate for the entered height to a distance of Z = [Entered height].IMAGE GOES HERE 
5. Insert the stub to the stage:
   1. Use a 1.5 mm allen wrench to carefully loosen the set screw, then insert the mount with the specimen to the stage, and tighten the set screw (Figure 5).
   2. Important: Ensure that stub (+/- adaptor) is seated fully into the stage mount. Use caution when loading samples to prevent accidental collision with detectors. Gloves should be worn when handling any components/sample material that goes into the vacuum chamber. IMAGE GOES HERE Skin oil and loose debris can damage the detector. Stubs should be prepared with gloves. For thin-sections and grain mounts, they should be wiped with isopropanol. It may be beneficial to leave a grain mount overnight in a vacuum or dessicator cabinet, as in particular, grain mounts can degas water and other volatiles.
   3. If you lose the set-screw, there are a few spares in a small ziploc bag in the clear case with SEC SEM accessories.
6. Capture the specimen image to aid in navigation of the stub. To do so:
   1. Slide the door halfway closed to the point where the door catches and clicks.
   2. In Nanoeye, click the “Camera” button and the screen display will show the specimen (Figure 6). 
   3. With the camera activated, right click the “Camera” button to activate the brightness/contrast menu (Figure 6).
      Click the camera button again to take an image.IMAGE GOES HERE 
7. Close the chamber door gently and push the Exchange button (Figure 1.3) to put the chamber under vacuum. Gently press the door into the SEM to aid the seal as the vacuum begins to pump down.

B. Turning on the beam

1. Once the machine is under vacuum (indicated by the SEM beeping twice), navigate to the Operation Panel (Figure 7). IMAGE GOES HERE 

2. Select an accelerating voltage (range: 1 to 30 kV; see table 1 for working guide).

   1-5 kV	Delicate or uncoated samples (e.g. microfossils)
   5-10 kV	Coated biological samples (e.g. Au coated microfossils, recommended)
   10-30 kV	Carbon-coated thin section samples

   Table 1. Working guide for accelerating voltage

3. Select which detector to use:
   1. the secondary electron (SE) detector, which returns an image of the topography of the sample's surface and is generally used for micropaleontological identification with SEM stubs
   2. or the back-scattered electron (BSE) detector, which returns a value based on the average atomic number (Z) of a given material and is generally used on thin sections or grain mounts.
   3. User guide can have a more in-depth explanation of the two different detectors and show samples of SE and BSE images.
4. Click the START button to generate an electron beam. 
5. Monitor the emission current, which should be around 110uA. If it has diverged by more than ±20 uA, please notify a technician as this either means the filament is about to die or the beam is not stable, and image quality will be subpar. how to change the filament

C. Image refinement

Basic software controls

1. Once the beam is on, you can navigate around the sample in the x-y direction by double-clicking on the SEM display or sample map/camera screen. Can also change the x-y.
2. Cautionary note about changing the z, rotation
3. Magnification can be adjusted using the mouse wheel.

Focusing the microscope

1. Click the buttons under “Focus” area to adjust the focus (Figure 10) until an image is visible. This should be at approximately _______________. <<<need to describe how to use the UI
2. In several steps, increase the magnification then adjust the Focus first using the Coarse Focus Adjustment arrows, then the Fine Focus Adjustment arrows. Do this until you have reached a good focus at the desired magnification.

Tips for focusing the microscope

1. Tips.....
2. If the screen wobbles significantly in either horizontal or vertical direction during focus adjustment, this indicates that the variable aperture strip may be out of alignment. Let a technician know and they will manually adjust the aperture’s alignment using knobs on the side of the instrument. Include instructions

Improving the image

Tighten the beam area (spot size)

1. If the image looks dark, adjust the “Spot Size” (the lower the % value, the greater the amount of beam and the brighter the image). 
2. Reduce the spot size of the beam when the amount of focused beam becomes larger. This option is suitable for ultra-fine images (the higher the % value, the tighter the beam focus, but the image will also become darker).

Adjust the brightness/contrast

Change the scan speed

Get a technician

Advanced image correction (for technicians)

Correct the beam shape

Align the strip aperture

Adjust the stigmation

Align the beam

Recommended settings

Additional tips

• Dealing with movement
  
  • Sea state
  • Jostling the table
• For high magnification (>5000x; unlikely at sea conditions)
  • Change the variable strip aperture
  • Increase the Spot Size (higher number)
  • Change the accelerating voltage up or down
  • Adjust the stigmation
  • Focus the microscope/decrease the scan speed

If the sample is charging

An explanation of what charging means and why it's bad

• Make the spot size more diffuse
• Decrease the accelerating voltage
• Try Low Vacuum
• Make sure the sample is conductive

D. Capture an image

Saving the image

Annotating the image (optional)

E. Shutdown

1. When you are finished scanning, turn the beam off by clicking the Stop button in the Operation dropdown panel.
2. Bring the motor back to the zero position by clicking Home
3. Nanoeye software can now be closed
4. Push the vacuum button (Figure 1.3). This will initiate the chamber filling with air, and you can track the progress using the LED strip on the front of the instrument.
5. The door will be easily opened once pumped with air
6. Remove your specimen mount from the holder (Figure 5), removing the specimen from the stub adaptor if used.
7. Close the door, then push the vacuum button (Figure 1. 3) to initiate a vacuum sequence.
8. Once the vacuum has be reached, turn off the SEM (Figure 1.2). When not in use, the SEM should be closed, at vacuum, and powered off.

III. Uploading Data to LIMS

Updated SEM Uploader

IV. Credits

Version 1 of this document was written by Luan Heywood, Sarah Kachovich and Kara Vadman, on Exp 397T. This document was reviewed by ___ on ___.

V. Archived Versions